Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
1. The Field of the Invention
This invention relates to the medical arts, particularly to the field of transgenics and gene therapy. The invention is particularly directed to in vitro and in vivo methods for genetically modifying male germ cells and support cells (i.e., Leydig and Sertoli cells), which methods incorporate a method of incorporating exogenous genetic material into the genome of a vertebrate to produce transgenic vertebrates and transgenic vertebrate animal lines.
2. Discussion of the Related Art
The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression, and interaction. This technology has been used to produce models for various diseases in humans and other animals. Transgenic technology is amongst the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function.
It is also used to study the relationship between genes and diseases. About 5,000 diseases are caused by a single genetic defect. More commonly, other diseases are the result of complex interactions between one or more genes and environmental agents, such as viruses or carcinogens. The understanding of such interactions is of prime importance for the development of therapies, such as gene therapy and drug therapies, and also treatments such as organ transplantation. Such treatments compensate for functional deficiencies and/or may eliminate undesirable functions expressed in an organism.
Transgenesis has also been used for the improvement of livestock, and for the large scale production of biologically active pharmaceuticals. Historically, transgenic animals have been produced almost exclusively by microinjection of the fertilized egg. The pronuclei of fertilized eggs are microinjected in vitro with foreign, i.e., xenogeneic or allogeneic DNA or hybrid DNA molecules. The microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female. (E.g., P. J. A. Krimpenfort et al., Transgenic mice depleted in mature T-cells and methods for making transgenic mice, U.S. Pat. Nos. 5,175,384 and 5,434,340; P. J. A. Krimpenfort et al., Transgenic mice depleted in mature lymphocytic cell-type, U.S. Pat. No. 5,591,669).
This widely used technique requires large numbers of fertilized eggs, equipment to handle embryos and the facility to microinject them in vitro. This is partly because there is a high rate of egg loss due to lysis during microinjection. Moreover manipulated embryos are less likely to implant and survive in utero. These factors contribute to the technique's extremely low efficiency. Superovulated mammals (e.g., primates, cows, horses, pigs, and mice) produce only 10-20 or less eggs per female animal per cycle, even after hormonal stimulation, and only 1% of microinjected mouse eggs (Palmiter, R. D. and Brinster, R. L., Germline transformation of mice, Annu. Rev. Genet. 20:465-99 [1986]), and 0.1% of cattle, sheep and pig eggs (Wall, R. J., et al., Making transgenic livestock: genetic engineering on a large scale, J. Cell Biochem. 49:113-120 [1992]) develop into transgenic animals. Typically, 300-500 fertilized eggs must be microinjected to produce perhaps three transgenic animals. Consequently, generating large animals with these techniques is prohibitively expensive. For this reason, mammalian transgenic technology has been confined almost exclusively to mice due to their high fecundity. Little has been done to improve the generation of transgenic animals by the microinjection of a transgene into fertilized eggs (Gordon, J. and Ruddle, F. H., Integration and stable germ line transmission of genes injected into mouse pronuclei, Science 214:1244-1246 [1981]).
While small animals such as mice have proved to be suitable models for certain diseases, their value in this respect is limited. Larger transgenic animals would be much more suitable than mice for the study of the effects and treatment of most human diseases because of their greater similarity to humans in many aspects, and better for studying organ systems or behavior. Larger mammals are also more suitable than mice as potential organ donors to humans due to the comparable size of their organs. Now that transgenic animals with the potential for human xenotransplantation are being developed, more of these larger animals will be required. Transgenic technology will allow that such donor animals will be immunocompatible with the human recipient.
In contrast to only 10-20 eggs per female even after treatment with superovulatory drugs, most male mammals, including mice and nearly all larger mammals, generally produce at least about 108 spermatozoa (male germ cells) in each ejaculate. For this reason alone, male germ cells will be a better target for introducing foreign DNA into the germ line, leading to the generation of transgenic animals with increased efficiency and after simple, natural mating.
Nevertheless, attempts to generate transgenic mice using spermatozoa to carry DNA into the egg (Lavitrano, M., et al., Sperm cells as vectors for introduction of DNA into eggs: genetic transformation of mice, Cell 57: 717-723 [1989]; WO-A-90/08192), have not been validated (Brinster, R. L., et al., No simple solution for making transgenics, Cell 59:239-241 [1989]). Recently, transgenic mice were produced after the injection of exogenous DNA together with sperm heads into oocytes (Perry, A. C., et al., Mammalian transgenesis by intracytoplasmic sperm injection, Science 284:1180-1183 [1999]). Following uterine transfer, 20% of these embryos developed into transgenic offspring.
Genetic information has been transferred to embryos using retroviral vectors (Jaenisch, R., Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus, Proc. Natl. Acad. Sci. USA 73:1260-1264 [1976]), but the animals were mosaics with different gene insertions in different tissues. (Jaenisch, R., Retroviruses and embryogenesis: microinjection of Moloney leukemia virus into midgestation mouse embryos, Cell 19:181-188 [1980]). Recently, five transgenic calves were produced by injection of a pseudotyped replication-deficient vector based on the Moloney murine leukemia virus. The vector was introduced into the perivitelline space of metaphase II oocytes (Chan, A. W., et al., Transgenic cattle produced by reverse-transcribed gene transfer in oocytes, Proc. Natl. Acad. Sci. USA 95:14028-14033 [1998]).
An alternative, not yet fully realized, is the stable transfection of male germ cells in vitro and their transfer to a recipient testis. Transfer of genetically marked germ cells to the testis yielded offspring, but so far no transgenic progeny have been produced (Brinster, R. L. and Avarbok, M. R., Germline transmission of donor haplotype following spermatogonial transplantation, Proc. Natl. Acad. Sci. USA 91:11303-11307 [1994]).
Spermatogenesis is the process by which a diploid spermatogonial stem cell provides daughter cells which undergo dramatic and distinct morphological changes to become self-propelling haploid cells (male gametes) capable, when fully mature, of fertilizing an ovum.
Primordial germ cells are first seen in the endodermal yolk sac epithelium at E8 and are thought to arise from the embryonic ectoderm (A. McLaren and M. Buehr, Cell Diff. Dev. 31:185 [1992]; Y. Matsui et al., Nature 353:750 [1991]). They migrate from the yolk sac epithelium through the hindgut endoderm to the genital ridges and proliferate through mitotic division to populate the testis.
At sexual maturity the spermatogonium goes through 5 or 6 mitotic divisions before it enters meiosis. The primitive spermatogonial stem cells (A0/As) proliferate and form a population of intermediate spermatogonia types Apr, Aal, A1-4 after which they differentiate into type B spermatogonia. The type B spermatogonia differentiate to form primary spermatocytes which enter a prolonged meiotic prophase during which homologous chromosomes pair and recombine. The states of meiosis that are morphologically distinguishable are; preleptotene, leptotene, zygotene, and pachytene; secondary spermatocytes and the haploid spermatids are later stages. Spermatids undergo great morphological changes during spermatogenesis, such as reshaping the nucleus, formation of the acrosome and assembly of the tail (A. R. Bellve et al., Recovery, capacitation, acrosome reaction, and fractionation of sperm, Methods Enzymol. 225:113-36 [1993]). The spermatocytes and spermatids establish vital contacts with the Sertoli cells through unique hemi-junctional attachments with the Sertoli cell membrane. The final changes in the maturing spermatozoan (i.e., spermatozoon) take place in the genital tract of the female prior to fertilization.
Initially, attempts were made to produce transgenic animals by adding DNA to spermatozoa which were then used to fertilize mouse eggs in vitro. The fertilized eggs were then transferred to pseudopregnant foster females, and of the pups born, 30% were reported to be transgenic and express the transgene. Despite repeated efforts by others, however, this experiment could not be reproduced and no transgenic pups were obtained. Indeed, there remains little doubt that the transgenic animals reputed to have been obtained by this method were not transgenic at all and the DNA incorporation reported was mere experimental artifact. Data collected from laboratories around the world engaged in testing this method showed that no transgenics were obtained from a total of 890 pups generated.
In summary, it is currently possible to produce live transgenic progeny but the available methods are costly and extremely inefficient. Spermatogenic transfection in accordance with this invention, either in vitro or in vivo, provides a simple, less costly and less invasive method of producing transgenic animals and one that is potentially highly effective in transferring allogeneic as well as xenogeneic genes into the animal's germ cells.
To facilitate in vitro transfection of male germ cells and implantation into a testis of a recipient male vertebrate it is advantageous first to depopulate the testis of the recipient vertebrate of untransfected male germ cells before transferring transfected male germ cells into it.
Depopulation of testis has commonly been done by exposing the whole vertebrate to gamma irradiation (X-ray), or localizing irradiation to the testis. (E.g., G. Pinon-Lataillade et al., Endocrinological and histological changes induced by continuous low dose gamma-irradiation of rat testis, Acta Endocrinol. (Copenh) 109(4): 558-62 [1985]; G. Pinon-Lataillade and J. Maas, Continuous gamma-irradiation of rats: dose-rate effect on loss and recovery of spermatogenesis, Strahlentherapie 161(7):421-26 [1985]; C. R. Hopkinson et al., The effect of local testicular irradiation on testicular histology and plasma hormone levels in the male rat, Acta Endocrinol. (Copenh) 87(2):413-23 [1978]; G. Pinon-Lataillade et al., Influence of germ cells upon Sertoli cells during continuous low-dose rate gamma-irradiation of adult rats, Mol. Cell. Endocrinol. 58(1):51-63 [1988]; P. Kamtchouing et al., Effect of continuous low-dose rate gamma-irradiation on rat Sertoli cell function, Reprod. Nutr. Dev. 28(4B):1009-17 [1988]; C. Pineau et al., Assessment of testicular function after acute and chronic irradiation: further evidence for influence of late spermatids on Sertoli cell function in the adult rat, Endocrinol. 124(6):2720-28 [1989]; M. Kangasniemi et al., Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes, Anat. Rec. 227(1):32-36 [1990]; G. Pinon-Lataillade et al., Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions, Reprod. Nutr. Dev. 31(6):617-29 [1991]).
The mechanism of gamma radiation-induced spermatogonial degeneration is thought to be related to the process of apoptosis. (M. Hasegawa et al., Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in p53-deficient mice, Radiat. Res. 149:263-70 [1998]).
Another method of depopulating a vertebrate testis is by administering a composition containing an alkylating agent, such as busulfan (Myleran). (E.g., F. X. Jiang, Behaviour of spermatogonia following recovery from busulfan treatment in the rat, Anat. Embryol. 198(1):53-61 [1998]; L. D. Russell and R. L. Brinster, Ultrastructural observations of spermatogenesis following transplantation of rat testis cells into mouse seminiferous tubules, J. Androl. 17(6):615-27 [1996]; N. Boujrad et al., Evolution of somatic and germ cell populations after busulfan treatment in utero or neonatal cryptochidism in the rat, Andrologia 27(4):223-28 [1995]; R. E. Linder et al., Endpoint of spermatotoxicity in the rat after short duration exposures to fourteen reproductive toxicants, Reprod. Toxicol. 6(6):491-505 [1992]; F. Kasuga and M. Takahashi, The endocrine function of rat gonads with reduced number of germ cells following busulfan treatment, Endocrinol. Jpn 33(1):105-15 [1986]).
Cytotoxic alkylating agents, such as busulfan, chlorambucil, cyclophosphamide, melphalan, or ethyl ethanesulfonic acid, are frequently used to kill malignant cells in cancer chemotherapy. (E.g., Andersson et al., Parenteral busulfan for treatment of malignant disease, U.S. Pat. Nos. 5,559,148 and 5,430,057; Stratford et al., Stimulation of stem cell growth by the bryostatins, U.S. Pat. No. 5,358,711; Luck et al., Treatment employing vasoconstrictive substances in combination with cytotoxic agents for introduction into cellular lesion, U.S. Pat. No. 4,978,332). Treatment of mice with busulfan (13 mg-40 mg/kg body wt.), was reported to deplete male germs cells in the testis; both stems cells and differentiating spermatogonia were killed; doses over 30 mg/kg body weight resulted in azoospermia for up to 56 days after treatment. (L. R. Bucci and M. L. Meistrich, Effects of busulfan on murine spennatogenesis: cytotoxicity, sterility, sperm abnormalities and dominant lethal mutations, Radiation Research 176:259-68 [1987]).
The present invention addresses the need for spermatogenic genetic modification, either in vitro or in vivo, that is highly effective in transferring allogeneic as well as xenogeneic genes into the animal's germ cells and in producing transgenic vertebrate animals. The present technology addresses the requirements of germ line and stem cell line gene therapies in humans and other vertebrate species, including the need for a superior method of depopulating a testis of untransfected male germ cells. The present technology is of great value in producing transgenic animals in large species as well as for repairing genetic defects that lead to male infertility. Male germ cells that have stably integrated the DNA are selectable. These and other benefits and features of the present invention are described herein.